RESPIRATORY STUDIES OF THE MICROCOCCI RESPIRATORY STUDIES OF THE MICROCOCCI by Tom Duvall Nunheimer A THESIS Submitted to the Graduate School of* Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology 1941 ProQuest Number: 10008397 All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008397 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 ACKNOWLEDGMENT The author wishes to express his sincere appreciation to Dr. F* W. Fabian, Professor of Bacteriology, under whose able guidance this work was done, for his unfailing interest through­ out the course of the work and for his interest and criticisms during the preparation of this manuscript* The author also wishes to express his sincere gratitude to Professor 0. D. Ball of the Department of Chemistry for many helpful suggestio: made throughout the course of the experiment* Table of Contents Introduction — ---- — --- i Study of Dehydrogenase Activity Literature ---- — ------------- — Method ------------------------------ 2 4 Results —----------- *--------Discussion--------------------- 7 11 Study of the CompleteRespiratory Mechanism Method--------------------Results--------------- — 15 — 15 Discussion--- -----------------------17 Study of Respiratory Inhibitors Influence of Inhibitors upon Dehydrogenases Literature — MethoB------------Results------- -- ------------ ----— Discussion-------Influence of Inhibitors upon Oxygen Uptake Literature— — •— ------Method-----Results ---------Discussion --------------- — ---- — — ^0 52 55 56 57 5S 42 45 Examination of Micrococci for Presence of Ather Enzymes Polyphenol oxidase ----- *---------- 54 Catalase---*------------------------- 55 Cytochromeoxidase------------------Study of the BActerial Pigment — --------- — — 56 - 58 Summary--------------------------- 62 Bibliography 65 Introduction The respiration of bacteria is a subject of primary importance in understanding the mechanism by which energy is furnished to the cell* These energy mechanisms in turn form an hypothesis which enables a better understanding of the methods by which foods are made available for life processes* Respiratory studies likewise assist in an understanding of many biochemical reactions such as one finds in the oxidation-reduction of many indicators and dyes used in bacteriology such as methylene blue, litmus, dyes used in Endo’s medium, indicator in tellurite medium, eosin-methylene blue and many others* The sensitivity of gram-positive and gram-negative organisms to certain dyes such as brilliant green and crystal violet might indicate a fundamental difference in the enzymatic makeup of the organism* A study of the respiratory enzymes found in bacteria leads to a better understanding of the fundamental differences between aerobiosis and anaerobiosis, and has helped to explain the relation of oxidation to fermentation. In view of the fact that respiratory enzymes play an important part in many of the fundamental reactions of the cell and since there has been only a very limited amount of work done upon the respiratory enzymes of the Micrococci, the present study was undertaken* - 2- Literature Survey of the Dehydrogenases It is generally accepted that biological oxidationreduction manifests itself as a transfer of hydrogen (electrons) from a donator to an acceptor, the transfer yielding energy to the organism. The role of oxygen is that of a hydrogen acceptor, while under anaerobic conditions the oxygen is replaced by some other suitable acceptor. Metabolites such as glucose, succinic acid, ethyl alcohol, etc., are so changed under the influence of certain specific cellular agents that hydrogen atoms become transferred to reducible substances. An activation of the metabolite by the cell initiates the hydrogen transfer; the specific enzymes activating this hydrogen transfer being known as dehydrogenases• The presence of dehydrogenases is generally demonstrated by means of the Thunberg (1918) methylene blue technique. P revious to 1957 studies of the dehydrogenases produced by bacteria were limited almost entirely to the colon-typhoid group. ( Braun and Wordehoff 1955* Oook 1950, Green and Stickland 1954-a, 1954b, Quastel and Wooldridge 1928, Glass 1956, and Yudkin 1955*1954- Wooldridge, Knox and 1957) In 1952 Braun and Vasarhelyi studied the dehydrogenases of Staphylococcus aureus and found that relatively few substrates were dehydrogenated. Among the carbohydrates and related compounds fructose, maltose, sucrose and glycerol were oxidized while arabinose,galactose,lactose,mannito1 and dulcitol were not activated. - 3- Of thirteen organic acid salts used only formate, lactate, pyruvate and fumarate were actively dehydrogenated while such salts as succinate, tartrate, acetate, malate and citrate were inactive* Most of the amino acids were activated with the exception of glycine, valine, leucine and phenylalanine* Fabre (1955) studied the dehydrogenases produced by Staphylococcus aureus* Of the 67 substrates tested only 27 were found to be active* The most actively dehydrogenated were glucose, mannose, galactose, sucrose, lactose, xylose, maltose, lactate and formate* Substrates weakly activated were succinate, fumarate, ethyl alcohol and glutamate* The hydrogen donors which were inactive or doubtful were dulcitol, rhamnose, acetate, citrate, oxalate, alanine, phenylalanine, asparagine and aspartate. Ehrismann (1937) made a study of the dehydrogenases produced by micrococci and streptococci. The dehydrogenase activity was determined for Staphylococcus aureus and albus, Streptococcus pyogenes* Micrococcus tetragenus and candicans* Most of the substrates were found to be activated by all the organisms, the difference being quantitative rather than qualitative. The main exceptions were the inability of M* candicans to activate malate, ethyl alcohol and arabinose; the inability of M* tetragenus to activate dulcitol; and the ability of Staphylococcus albus to dehydrogenate asparagine. - 4- Experimental Study of* the Dehydrogenase Activity of the Micrococci The various strains of micrococci were tested by the Thunberg (19X8) technique to determine the dehydrogenase activity. Method The organisms were grown in Roux bottles at room temperature for JO to ^4 hours on a medium of the following composition: 0.5 per cent 0*2 per cent 0.2 per cent 0*2 per cent 5*0 per cent pH 7*0-7*l peptone yeast extract peptonized milk meat extract agar After incubation the growth was removed and washed three times (by centrifugation) with saline and suspended in saline in a bottle fitted with a tube for sterile aeration* The cell suspension was made up to a volume of ten milliliters for each Roux bottle in the harvest. The cell suspension was stored at five degrees and aerated at that temperature before use* In the Thunberg tube was placed 1 ml. substrate (M/lO unless otherwise indicated), 1 ml* saline, 1 ml. methylene blue 1-5000, and 1 ml. of phosphate buffer* placed 1 ml. of aerated cells* In the hollow stopper was - 5- The buffer used was a mixture of M/50 KeHP04 and M/50 KHgF04 with a pH of 6.85* The tubes were evacuated for 2.5 minutes with constant shaking and incubated in a water bath at 40°C. After allowing for equilibrium the cells were tipped into the reaction mixture and the time required to bring about complete reduction of the methylene blue was noted. The cell suspension was diluted with saline so that the reduction time, in the absence of added substrate, was about one hour. In this connection, Yudkin ( 1957) found that with Escherichia coli dilution of the cells resulted in a dispro­ portionate fall in the dehydrogenase activity. This was found to be due to the presence of a coenzyme which became the limiting factor at higher dilutions. In view of this fact, boiled cells were added to the diluted cell suspension to reduce the reduction time to the value calculable from the degree of dilution. The substrates were made up to M/lO unless otherwise indicated and adjusted to pH 7*1* The time required for complete reduction of the methylene blue was noted and the dehydrogenase values were calculated and reported in the following-manner: time of control —reduction -— —:-----r~rr-r— r— — reduction time with substrate „ ,, „ , x 100 = Dehydrogenase Value ° - 6- It can be seen that the reduction time of the control in which there was no added substrate would in each case result in a Dehydrogenase Value of 100, while a Dehydrogenase Value of 200 in the presence of a substrate would indicate that the reduction time was half that of the comtrol* Preliminary tests showed the dehydrogenase activity to be stable for three or four days if the cell suspensions were stored at five degrees centigrade* - Table 1* 7- The Rate of Dehydrogenation of Sugars and Alcohols by Micrococci. The Figures represent Dehydrogenase Values. •H 0 d d d 0 u '• ■ 1 h S3 M# m flavus CO •H --- « --- M.oinn©bareas m d-Arabinose 144 115 145 170 155 1-Arabinose 122 149 127 215 298 d-Xylose 127 150 155 516 425 d-Glucose 250 145 171 1260 2555 d-Fructose 529 176 155 971 1900 d-Mannose 210 185 124 744 1100 d-Galactose 125 159 297 919 516 Lactose 171 147 212 669 62 Maltose 1000 285 225 821 1100 Sucrose 567 579 156 956 1090 Raffinose 855 215 428 1075 1950 1-Rhamnose 145 169 111 1016 459 Ethyl Alcohol 550 758 6400 500 2655 Glycerol l4o 166 628 500 810 Dulcitol 161 200 102 225 2400 d-Mannitol 264 172 229 225 5900 - Tabl© 1, Continued. 8- The Rate of Dehydrogenation of Organic and Amino Acids by Micrococci. •rl ■A' ra CO pi 0 -p ;3 1— 1 *_- CQ PS Is 1— 1 pi 0 td •H P S3 Substrate U CQ S3 03 H «H 'i-i • M. aurantiaeus 09 i i Substsate M. luteus ---------0 •H O 09 d CD u 08 4* QQ CQ sd 0 a 0 as *> •cf as u £ 1—l 0 ES 1--- I ctS «H Td Pi id CD Pi PI •H O CD Pt as * ♦ • £3 56*5 27.7 31.3 6 5 .0 56.5 1-Arabinose 39.1 40.4 5 0 .5 64.1 55.9 d-Xylose 56.5 1 5 0 .5 52.5 74*5 65.4 d-Glucose 145*7 79.5 141.7 318.7 204.0 d-Fructose 67-9 4 0 .7 99*4 465.8 7 4 .1 d-MannoBe 62*9 4 0 .5 51.9 108.4 1 5 4 .6 d-Galactose 98*7 138.5 55.8 7 0 .2 60.8 Lactose 81.5 159.2 7.3 54.6 57.4 Maitose 145.8 193.4 41.1 371.5 180.0 Sucrose 201.8 145-2 540.5 421.2 164.1 Raffinose 58*9 1 6 5 .0 55*6 79.4 66.5 1—Rhamnose 55-5 147.5 25.1 46.8 150.0 194.8 333.1 406.7 552.5 Glycerol 46*5 151.4 279.6 378.5 2 5 0 .1 Dulcitol 95.0 112.5 18.7 45.5 46.7 d-Mannitol 62.6 45.5 71.3 415.7 57.6 Ethyl Alcohol * d-Arabinose ro •J t— CQ & O as •H 4~> Pi aS u P* 01 Substrate • S3 03 zt > aS 1— I -p—i - Table 5* Continued. The Rate 16- of Oxygen Uptake by Micrococci* •H & 0 03 03 PJ 03 +3 P* 03 > Pi a aJ *!“1 +3 Et cd •H <13 fs El 03 TO m si aS p—1 3 r*H * to a o etf •H £l Ctf Jh as • ro © as X2 © fl M. freundenr e i chii •H O • v M/10 1.4xl0~2 Glucose Sucrose Lactate Succinate Glycine d1-b-Phenylalanine - 51 - 28 - 10 - 5 Glucose Sucrose Lactate Succinate Glycine dl-b-Phenylalanine -+■ 18 - 7.2 - 8.8 + 25 Glucose Sucrose Lactate Succinate Glycine dl-b-Phenylalanine + - Glucose Sucrose Lactate Succinate Glycine dl-b-Phenylalanine + + - Glucose Sucrose Lactate Succinate Glycine dl-b-Phenyl­ alanine - M/100 M/1000 1.4xl0~5 1.4x10"^ + M/10.000 1.4xlo“5 50 22 16 15 - 51 - 58 + + + 7.1 11 6.4 28 - 59 - 72 59 46 26 56 + 25 - 5 - 6.9 - 81 - 66 55 24 11 11 - 24 + 55 + 51 - 50 - 5 18 18 24 51 - 5.4 - 7.8 - 58 - 59 - 80 -55- Table 5* Continued• The Per Cent Increase or Decrease in Dehydrogenase Activity in the Presence of Inhibitors. Gluco se Sucrose Lactate Succinate Glycine d1-b-Phenylalanine - 12 - 29 + 6 - 4o Glucose Sucrose Lactate Succinate Glycine dl-b-Phenyl­ alanine + + + - + 64 - 52 - 29 CO' • Glucose Sucrose Lactate Succinate Glycine dl-b-Phenylalanine M .cinnebareus Initial concentration Final concentration Sodium azide M/100 M/1000 1.4x10“^ 1.4x10”^ Glucose Sucrose Lactate Succinate Glycine dl-b-Phenylalanine - 59 + 26 +160 - 52 Glucose Sucrose Lactate Succinate Glycine dl-b-Phenylalanine + m M* fla v u s f | i 1 —t 03 P* O CO •H S3 c0 L:H i S3 *H © S3 rCj pi O © -H ?H © - M/10.000 1.4xl0"5 4 - 20 - 16 - 27 - 62 22 15 17 5-5 + 56 + 16 - 47 - 71 + 5.5 - 6.4 - 16 - 77 - 8 + 50 - 61 - 4o 5*7 - 55 - 58 - 60 - 60 - 60 -56- Discussion A great, difference -was found among "the organisms in regard to their sensitivity toward inhibitors (Table 5). The dehydrogenases of M* luteus and M* flavus were stimulated by a cyanide concentration as high as 1*4x10 molar while the other organisms were generally inhibited by this concentration. aurantiacus showed the greatest sensitivity toward cyanide, being inhibited by a concentration of 1*4x10”^ molar* The specific dehydrogenases were found to vary in their sensitivity to inhibitors since dehydrogenation of the amino acids was greatly inhibited by a cyanide concentration of 1.4x10 molar while this concentration did not affedt the other dehydrogenases* M. cinnebareus and M. flavus were the least sensitive to monochlor-acetate since they were generally stimulated by a concentration of 1.4x10—2 molar, while M. aurantiacus appeared to be the most sensitive toward sodium monochlor-acetate in this concentration* The amino acid dehydrogenases were more sensitive toward sodium monochlor-acetate than were the sugar and fatty acid dehydrogenases* Sodium azide appeared to be a stronger inhibitor than sodium cyanide since a concentration of 1.4xL0 ' molar sodium azide inhibited the dehydrogenases to a greater extent than did this concentration of sodium cyanide* -57- M* .flo-vus» however, was generally stimulated, by both sodium cyanide and sodium azide, and in this case the stimulation produced by azide was much less than that produced by cyanide* The glucose dehydrogenase of M. aurantiacus appeared to be excpetionally resistant to both sodium monochlor-acetate and sodium azide while the other dehydrogenases of this organism appeared to be very sensitive to all the inhibitors* The amino acid dehydrogenases were found to be uniformily more susceptible to all the inhibitors than were the other dehydrogenases produced by the organisms. Influence of Inhibitors upon Oxygen Uptake by Micrococci In 1924 Gallow studied the oxygen uptake of a number of bacteria and found very little oxygen uptake for Staphylococcus aureus and Sarcina aurantiaca* G-erard (1951) found that the Q0S ( c.mm.oxygen uptake per hour per mgm. dry weight of cells) for Sarcina lutea to be between four and six, while the Q0S in glucose was about 20. Methylene blue increased the oxygen uptake of endogenous respiration about 50 per cent while theoxidation of lactate was inhibited between 10 and ^>0 per cent. Cyanide in a concentration of M/100 didnot decrease the oxygen uptake of the cells in the presence of any of the substrates, while a concentration of M/lO inhibited respiration about 50 per cent. Working with the same strain one year later, however, Barron (Gerard,1951) found that M/lOO cyanide inhibited a respiration about 50 per cent. Oook, Haldane and Mapson (1951) found that cyanide concentrations below 2x10-5 molar did not bring about serious inactivation of the dehydrogenases of E. coli. With formate as the substrate it was found that cyanide strongly inhibited oxygen uptake, but that the addition of methylene blue together with cyanide resulted in an oxygen uptake nearly that of the formate alone in the absence of inhibitor. With lactate as the substrate methylene blue alone (m /250) inhibited respiration about 74 per cent, and when cyanide and methylene blue were added together only 67 per cent of the original uptake (lactate alone) was obtained. After studying the effect of the various inhibitors upon the dehydrogenases, the effect of the inhibitors upon the complete respiratory mechanism of the cell was determined* Method In the Warburg vessel were placed 1 ml.cells, 1 ml. buffer pH 7.05, 0.1 ml. saline, and 0.5 ml. inhibitor which was adjusted to pH 7*1. In the center cup was placed 0.2 ml. of twenty per cent NaOH to take up the carbon dioxide evolved. The buffer was a mixture of M/50 KsHP04 and M/50 KHsP04. When sodium monochlor-acetate and sodium azide were used as the inhibitors the cells, buffer, saline and inhibitor were allowed to stand, with frequent agitation, for ten minutes -39- before the addition of M/50 substrate. In those cases where cyanide was used as the inhibitor, the cyanide was allowed to remain in contact with the cells, buffer, and saline for twenty minutes before the addition of the substrate, and the carbon dioxide absorbent used was the KOH-KCN mixture suggested by Krebs (1955). After the addition of the substrate the vessels were placed in the water bath at 55°0* and ten minutes allowed for equilibrium. The oxygen uptake vislq followed for the next 60 minutes• Using glucose as a representative substrate it was found that M. luteus was resistant to cyanide. _2 a cyanide concentration of 1.4x10 about 69 per cent. Figure 2 indicates that molar inhibited respiration A concentration of 1.4x10—5 molar inhibited respiration slightly during the first 45 minutes, but this period was followed by an increase in rate so that the total oxygen uptake at the end of the 60 minutes was identical with the uptake in the absence of inhibitor. M.aurantiacus likewise showed such an increase with glucose as the substrate* Cyanide concentrations less than 1.4x10 y molar brought about a stimulation. This amounted to 45 per cent in a -4 cyanide concentration of 1.4x10 molar while half this concentration of cyanide stimulated 21 per cent. -4o- 1.4x10' 200 None 1.4x10' 120 80 Gubic mm oxygen uptake 160 15 60 Time in Minutes Figure 2* The Effect of Potassium Cyanide (in molar concentration) upon the Oxygen Uptake of M# luteus. -41- The influence of inhibitors upon the oxygen uptake of the organisms waa determined using as substrates glucose, sucrose, lactate, succinate, glycine and dl-b-phenylalanine• A concentration of inhibitor was chosen which did not exert too pronounced an effect upon the dehydrogenase and the effect of this concentration on the oxygen uptake of the cells was determined* The results are shown in Table 9 and are summarized in Tables 6,7 and 8. -42- Table 6. The Per Gent Increase or Decrease in Respiration in the Presence of Potassium Gyanide and Methylene Blue. Potassium cyanide Final molar concentration 1.4xl0“2 1.4x10"^ 1.4x10^ KON + o M .B . Glucose Sucrose Lactate Succinate -.70 - 77 - 79 - 95 - 45 15 74 94 1 mgm. - 5*5 +157 + 45 - 6.5 i—i CH ' ter —• ■ Glucose Sucrose Lactate Succinate - + - 58 55 71 90 1 mgm* +101 + 44 + 5.1 - 5*5 M* aurantiacus ra M. B . alone Glucose Sucrose Lactate Succinate + - 42 91 47 76 0.1 - mgm. 55 85 15 65 - 80 82 71 87 0.1 + - mgm. 51 55 57 75 75 0.4 0.5 + + + - mgm. 25 58 59 59 Q) rJ rH • m in ci oS 58 78 76 95 Glucose Sucrose Lactate Succinate Glucose Sucrose Lactate Succinate - 88 - 92 - 95 - 26 - 77 + 9*6 - 25 - 54 + - 65 51 si. ° The cyanide concentration used was that recorded in the column to the left, and the methylene blue concentration was that amount in mgm. indicated in the column to right. -45- Table 7* The Per Gent Increase or Decrease in Respiration in the Presence of* Sodium monochlor-acetate* Sodium monochlor--acetate Pinal motar concentration 1.4xl0~^ Glucose Sucrose Lactate Succinate Glycine + 97 + 5*6 - 26 - 19 M~ flavus Glucose Sucrose Lactate Succinate + 6 + 9.1 - 27 - 7.6 + 16 M. aurantiacus Glucose Sucrose Lactate Succinate M. luteus M. cinnebareus M*freundenreichii Glucose Sucrose Lactate Succinate dl-b-Phenylalanine Glucose Sucrose Lactate Succinate 1.4xlO~5 l.4xio"5 - 6.5 - 1.8 * 7.2 + 8 *9 + 9*2 - 50 - 44 - 54 + 4.7 + 5-2 - 28 + 8.5 0.4 -44- Table 8* The Per Gent. Increase or Decrease in Respiration in the Presence of Sodium Azide# Sodium azide Final molar concentration M# luteus M# flavus M• auranti acu s M. cinnebareus M ♦fr eund enreichii 1.4xlO”5 Glucose Sucrose Lactate Succinate Glycine +161 + 16 - 26 - 15 Glucose Sucrose Lactate Succinate + - Glucose Sucrose Lactate Succinate Glycine + 0.2 Glucose Sucrose Lactate Succinate Glucose Sucrose Lactate Succinate 1.4X10-4 1.4xl0~5 - 42 4 #6 16 9 7.4 - 5 .8 + 2.1 + 8.8 + 2.6 - 59 55 - 14 - IO + 4.1 - 2.8 - 54 0.4 -45- Discussion It. was found ( Table 6) ‘that, cyanide in a concentration of 1.4x10 molar greatly inhibited the oxygen uptake of the cells. In general the inhibition of glucose was les3 than for the other substrates. Since most of the amino acid dehydrogenases were so strongly inhibited by a cyanide concentration as low as 1.4x10—5 molar, the effect of cyanide upon the oxygen uptake was not deter­ mined. However, the dl-b-phenylalanine dehydrogenase of M. cinnebareus was an exception since it was inhibited only five per cent and the oxygen uptake only nine per cent by a 1.4x10 molar concentration of cyanide. The decrease in respiration in the presence of cyanide indicated that the cell carriers operative in normal respiration were inhibited. Therefore the ability of methylene blue to replace these natural carriers was determined. Influence of Methylene Blue upon Cyanide Inhibition The methods were the same as for the study of the inhibitors alone except that for this work the methylene blue was made up with the cyanide and the pH of the mixture was adjusted to 7*05* This mixture of cyanide and methylene blue was added to the cells, buffer and saline and allowed to stand for 20 minutes before the addition of the substrate. The oxygen uptake was compared -46- with that in the presence of methylene blue alone which was likewise allowed to remain in contact with the cells for 20 minutes before the addition of the substrate. The organisms varied in their sensitivity toward methylene blue (Table 6). M. luteus and M* flavus were stimulated by the presence of 1 milligram of methylene blue in the presence of all substrates except succinate. The other organisms were found to be inhibited by this dye concentration, although M. freundenreichii was stimulated by 0.5 milligram in the presence of all substrates except succinate. M. cinnebareus was still more sensitive to methylene blue since even 0.1 milligram exerted strong inhibitive action in the presence of all substrates except lactate with which there was stimulation. Methylene blue was not capable of replacing the natural cell carriers to any great extent* For most of the organisms the cyanide inhibition was lessened slightly by methylene blue in the presence of glucose, sucrose and lactate (Table 6)* The cyanide inhibition was completely removed by methylene blue only in the case of M. flavus with sucrose as the substrate and M. aurantiacus with glucose as the substrate. The difference shown in the susceptibility toward cyanide and in the ability of methylene blue to act as a carrier indicates that one single mechanism is not operative in the oxidation of the different substrates. -47- Sodium monochlor-acetate in a concentration of* i.4xl0 molar inhibited the oxidation of* all the substrates with the exception of glucose with which there was stimulation (Table 7)* In two cases sucrose and succinate were also stimulated. Although sodium azide (Table 8) was found to be a stronger inhibitor than sodium monochlor-acetate, the stimulation and inhibition paralleled, indicating the point of attack of both inhibitors to be the same* The action of the inhibitors indicates that only one mechanism is probably operative for the oxidation of glucose. It is stimulated by sodium monochlor-acetate and sodium azide and inhibited by sodium cyanide. The cyanide inhibition, however, is partially removed by the presence of methylene blue* Two mechanisms exist for the oxidation of sucrose. With M* luteus, M. flavus and M* freundenreichii the oxidative mechanism is not susceptible to sodium monochlor-acetate or sodium azide, is inhibited by sodium cyanide but is not to any great extent reactivated by methylene blue* This mechanism may be identical with the oxidative mechanism operative in the presence of glucose. M. aurantiacus and M. cinnebareus differ in that the oxidation of sucrose is susceptible to all inhibitors, but reactivation does not take place in the presence of methylene blue* The oxidation of lactate is sensitive to all inhibitors* The cyanide sensitivity is removed by methylene blue only in the case of M. luteus. M. flavus and M. cinnebareus * -48- Th© amino acid dehydrogenases were very susceptible to the inhibitors, being strongly inhibited by a concentration —5 as low as 1.4x10 molar, therefore the inhibition of oxygen uptake was not determined. The dl-b-phenylalanine dehydrogenase of* M* cinnebareus. however, was inhibited by only five per cent in this concentration of cyanide and sodium monochlor-acetate. The oxygen uptake in the presence of this amount of sodium cyanide was found to be nine per cent, while the uptake in the presence of sodium monochlor-acetate was stimulated 4.7 pe** cent* The response of a dehydrogenase to an inhibitor, and the inhibition or stimulation of oxygen uptake in the presence of that inhibitor frequently did not parallel. This can be explained by the fact that respiration is the sum total of individual reactions. If the dehydrogenase activity limits the respiratory rate, any inhibition of the dehydrogenase will decrease the oxygen uptake (respiratory rate) to the same extent. If the dehydrogenase activity does not limit the respiratory rate, the dehydrogenase may be inhibited to a certain extent and still not decrease the rate of oxygen uptake* However, if the dehydrogenase and another enzyme is inhibited, the oxyegn uptake may or may not parallel the dehydrogenase activity depending upon which enzyme or reaction becomes the limiting factor. -49- Tab 1© 9« Th© Influence of Inhibitors upon Oxygen Uptake* The figures represent the c.mm. 0S uptake per mgm* nitrogen* Micrococcus luteus Substrate Inhibitor Glucose None CHgOlCOoNa NaN3 KCN ° KON + M*B. M. B. Sucrose None OHoClCOoNa kJ 3 KON ° KON + MB. M. B. Lactate None 0Hs0100sNa NaN3 KON ° KCN + MB. M. B. None CHoClOOoNa NaN3 Succinate KON ° KON + M.B. M. B. Glycine Final molar Concentration 15 22.2 1.4x10“!: 41.2 46.6 1.4x10“^ 5.0 l.4x10 16.2 1 mgm. MB. 1 mgm.. ___ 39.8 1.4x10“!: 1.4x10 $ l.4xlo“^ 1 mgm. MB • 1 mgm. 55.2 51.4 58.7 10.1 46.9 137^8 __ i.4xio“!: i.4xio“j i.4xio“^ 1 mgm. MB. 1 mgm* 47.9 37.5 55.0 12.6 17.1 105.1 1.4xl0”| 1.4xl0“j 1.4x10“ 1 mgm. MB. 1 mgm. 151.9 115.1 115.0 6.7 12.9 1 2 6 .6 None 1.4xlCf5 OHoOlOOoNa 1.4x10 j: hJ 3 1.4x10 KON 1 mgm. MB. ° KON + M.B. 1 mgm. M. B. ° 1 mgm* of methylene blue + given immediately above* Time in minutes 45 _30 60 40.7 81.1 94.0 10.3 25.9 61.2 56*0 115.6 139.9 15.6 54.2 66.8 72.6 145.2 189.5 21.4 40.1 74.2 102.8 102.8 110.5 20.2 88.2 223.8 146.2 146.6 157.6 124.0 269.5 185.0 192.1 214.4 41.8 I6 O .3 475. 1 98.7 75.8 71-4 21.5 174.2 148.7 110.1 105.7 31-7 41.9 235.7 191*5 142.2 159.5 40.9 50.1 278.2 574.8 259*5 244.5 14.8 22.3 349.8 463.7 575.5 585*7 22.1 28*5 426.9 620.9 501.9 526.7 33*7 35.9 580.8 3 0 .0 5 0 .2 163.2 220.7 551*5 328.6 252.0 164.8 2 0 3 .1 99.0 148.9 204.1 147.2 98.5 6 7 .0 58.0 46.9 180.4 1 9 6 .1 149.7 that concentration of cyanide 78.6 80.6 50.9 50.4 30.7 95.8 -50- Table 9* Continued. The Influence of Inhibitors upon Oxygen Uptake* The figures represent the c. mm* 0S uptake per mgm* nitrogen* Micrococcus flavus Inhibitor Final molar concentration Glucose None OHgOlCOgNa NaN3 KON KON + M.B. M. B. 1*4x10 % 1*4x10“^ 1.4x10 1 mgm. MB. 1 mgm. 16.2 24*7 17.1 8.7 15.9 47.1 52.5 59.5 52*1 17.7 24.0 81.0 47.2 55.6 56.1 27.8 51*2 106*7 61.0 64.8 58.2 57.6 57.6 122.5 Sucrose None CHoClCOoNa NaNa KON 0 KON + M.B. M. B. l.4xio~i: 1.4x10“^ 1.4x10"** 1 mgm. MB. 1 mgm. 45.9 40.6 49.0 7.5 49.5 55.2 85.5 74.2 98.4 16.2 105.4 115.6 122.2 144.2 145.9 24.5 155.8 156.0 157.6 171.9 182.5 55.2 1.4x10"! 1.4x10*5 1.4x10 1 mgm. M.B. 1 mgm. 66.6 54.4 42.4 11.1 18.1 84.0 99.0 68.6 82.2 19.4 29.0 115.9 141.8 100.8 125.7 108.6 104.2 105.7 8.9 14.9 265.0 215.8 215.5 19.6 28.5 179.9 455.5 529.5 None OHgOlOOgNa Lactate 0 KON KCN + M.B. M. B. None OHgOlOOgNa NaN5 Succinate KON 0 KON + M.B. M. B. 1.4x10"? i .4kio "5 1.4x10 1 mgm. MB* 1 mgm. 15 6 7 .8 Time in minutes 50 45 5 1 .0 45.2 154.6 60 2 0 9 .2 227.1 177.8 150.4 161.6 42.4 51.6 186.8 5 8 .0 468.2 452.8 452.7 57.2 47.7 511.5 4 5 2 .9 5 2 1 .8 28.2 ° 1 mgm* of methylene blue + that concentration of cyanide given immediately above* -51- Table 9* Continued. The Influence of Inhibitors upon Oxygen Uptake. The figures represent the c.mm. 0S uptake per mgm* nitrogen* Micrococcus aurantiacus Substrate Inhibitor Final molar concentration 15 Time in minutes 50 45 60 None_ CHgClCOgNa NaNs ' KON O KON + MB* M* B. — o 1.4x10 ^ l.4xio"? 1.4x10 0.1 mgm.MB* 0.1 mgm. 59.1 44.8 41.5 52.9 54.1 29.7 81.7 95*9 8 5 .I 75.5 115.9 54.5 121.7 141.7 122.0 112.1 171.7 84.6 165*7 190.3 164.0 187.0 252.9 110.2 None CHgClCOgNa NaN3 KCN o KON + MB. M. B. 117.6 121.8 115-5 104.6 9.9 29.1 254.5 257.1 227.9 201.1 19.0 47.4 525.2 51S. 7 590.7 1.4x10? 1.4xl0~2, 1.4x10 0.1 mgm.MB. 0.1 mgm. 5 1 2 .6 576.0 365.6 56.2 65.8 None CHgClCOgNa NaN3 KON KON + M.B. M. B. 1.4x10“^ 1.4x10"? 1.4x10 0.1 mgm.MB. 0.1 mgm. 68.5 60.2 65.8 55.8 151.4 117.0 128.7 2 5 .8 55.4 95-6 185.4 160.6 89.4 149.7 None OHgOlOOgNa NaN3 Succinate KCN o KON + M.B. M. B. 1.4x10 ? l.4xio”d 1.4x10 0.1 mgm.MB. 0.1 mgm. 64.0 68.1 64.1 45.8 14.4 26.9 126.5 159.6 128.2 96.8 29.5 44.8 191.1 209-1 194.1 148.4 44.2 51.6 248.6 270.8 255.9 59.0 45.0 82.6 88.4 126.8 156.6 176.4 192.0 Glucose S kju M vAv Dw T a - i. XiS-C uSLX© O Glycine None NaN3 _a 1.4x10 44.4 111.1 270.7 26.9 59.5 187-5 1 6 9 .2 5 8 5 .6 242.7 225.2 241.7 212.9 127*8 205.2 2 0 6 .2 6 0 .1 92.1 ° 0.1 mgm. of methylene blue + that concentration of cyanide given immediately above. -52- Table 9. Continued. The Influence of Inhibitors upon Oxygen Uptake. The figures represent the c.mm. 0S uptake per mgm. nitrogen. Microcoecus cinnebareus Substrate Glucose Sucrose Lactate Inhibitor Final molar concentrationL None CHoClCOoNa NaN3 KON ° KCN + M.B. M. B. 1.4xl0~? 1.4x10 p i.4xio"2 0.1 mgm.MB. 0.1 mgm. None CHgClCOsNa NaN3 KCN ° KON + M.B. M. B. 1.4xl0~2 1.4x10"' 1.4x10 0.1 mgm.MB. 0.1 mgm. None CHsClC02Na NaN3 KCN ° KCN + M.B. M. B. 64.6 7 2 .1 65.1 11.5 18.5 58.6 Time in minutes 50 45 159.0 155.8 159.8 17.0 5 0 .1 75-1 277.1 515.6 157.2 192.4 24.6 56.7 204.5 159.8 96.7 7 2 .6 1 5 0 .8 240.0 150.8 I6 6 .5 4.6 12.2 45.7 16.1 29.6 94.9 40.2 147.1 85.9 1.4xl0"2 50.7 7 0 .8 1.4x10^ l.4xio"“ 6.9 0.1 mgm. MB. 2 6 .5 0.1 mgm. 155.9 180.9 107.6 157.2 52.5 292.7 65.9 45.5 60.4 55.5 18.1 28.6 142*5 89.5 128.8 111.1 28.6 52.5 104.9 106.0 98.5 1 5 .8 222.5 228.8 207.3 25.8 14.5 2 5 .0 1,4x10”? 1.4xio”7 1.4x10 0.1 mgm.MB. 0.1 mgm. None CHgClCOgNa KCN ° KCN + M.B. M. B. i.4xio"1.4x10 0.1 mgm.MB. 0.1 mgm. 1 6 .1 60 204.0 229.0 211.4 24.1 42.6 106.2 77*1 55.5 None CHgClCOgNa NaN3 Succinate KCN ° KCN + M.B. M. B. dl-b— phenyl­ alanine 15 1 6 .6 2 6 6 .S 154.8 189*9 21.1 79.0 450.6 5 0 2 .6 284.4 52.1 54.6 1 5 6 .5 555.8 200.0 251.1 26.2 104.6 557.5 180.9 57.7 72.9 542.5 157.9 292.7 252.8 44.2 92.5 541.8 554.4 297-9 55.5 28.7 464.5 486.5 421.5 40.2 54.4 255*4 125.7 2 0 7 .2 ° 0.1 mgm. of methylene blue + that concentration of cyanide given immediately above. -55- Table 9* Continued* The Influence of Inhibitors upon Oxygen Uptake* The figures represent the c. mm. 0S uptake per mgm. nitrogen* Micrococcus freundenreichii Substrate Glucose Sucrose Inhibitor Final molar concentration None CH9 ClC0sNa NaN3 KCN ° KCN + M. B. M. B. — p 1.4x10 ^ 1.4x10 J 1.4x10 0.5 mgm.MB. 0.5 mgm. 65-7 74.5 59*6 15.5 1S .1 8 5 .I None CHgClCOgNa NaN3 KCN 0 KON + M. B. M* B. 1.4xl0“| 1.4xl0“i 1.4x10 0.5 mgm.MB* 0 .5 mgm. 62.5 65.1 62.4 51.4 55.4 89.5 None CHsClC0sNa NaN3 KCN 0 KON + M. B. M. B. 1.4x10 0.5 mgm.MB* 0.5 mgm. None CHgClCOgNa NaN3 Succinate KCN 0 KCN + M. B* M. B. 1.4x10“? l.4xio“7 1.4x10 0.5 mgm.MB. 0.5 mgm. Lactate 15 i .4x k Q: 1 .4xio~7 Time in minutes 45 30 _ 175.3 155.2 117.8 2 6 .6 54.7 168.4 1 2 1 .8 124.0 125.2 1 8 5 .2 168.4 56.9 49.4 2 2 1 .0 174.6 175.7 60 215.7 220.5 216.5 48.4 58.5 268.4 2 2 6 .2 172.9 182.6 174.4 251.9 225.5 255.5 248.0 227.1 512.5 1 0 0 .1 6 2 .5 9 4 .6 5 4 .9 5 9 .5 1 5 6 .1 188.1 146.7 179-7 117.7 72.8 265.6 265.4 194.1 257.5 186.7 100.8 585-1 533.5 258.7 524.0 249.2 122.8 465.8 1 9 .2 5 6 .I 21.8 12.5 58.9 24.0 15.1 21.6 17.4 50.5 54.7 33.6 21.5 27.5 22.9 8 .5 12.2 12.8 1 1 6 .8 1 1 2 .0 1 8 0 .0 65.7 6 9 .I 41.8 29.3 51.3 25.8 ° 0*5 mgm, of methylene blue + that concentration of cyanide given immediately above. -54- Examination of Micrococci for the Presence of O-fcher Respiratory Enzymes Examination for the Presence of Polyphenol Oxidase Polyphenol oxidase is specific in that it will oxidize the ortho di—hydroxy phenols such as catechol and pyrogallol, yet is practically inert against the meta- and para-hydroxy phenols* The oxidation of the former is used as a basis for the determination of polyphenol oxidase. determined at ^5°G The oxygen uptake by the cells was in the presence of 5 milligrams of catechol. In the Warburg vessel were placed 1 ml. cells, 1 ml. M/50 phosphate buffer pH 7*05, 1.1 ml. saline and in the center cup was placed 0.2 ml. of twenty per cent NaOH to take up the carbon dioxide evolved. Five milligrams of catechol in 0.5 ml. of water was placed in the side arm and added at equilibrium. The oxygen uptake was followed for 60 minutes and compared with the oxygen uptake of the cells in the absence of catechol. The results are shown in the following table. Table 10* The Polyphenol Oxidase Activity of Micrococci Organi sm M. luteus M. flavus M. aurantiacus M. cinnebareus M. freundenreichii q02 in absence of catechol qOs in presence of catechol 50.4 25.5 26.6 59.9 116.8 47.6 58.1 185.4 58.0 53.9 -55- Examination for the Presence of Catalase The manometric method similar to that introduced by Blaschko (1955) was used* This method makes use of the fact that catalase breaks down hydrogen peroxide to water and oxygen. Catalase can be determined, then, by measuring the oxygen given off from peroxide* A blank must be run to determine the oxygen uptake by the cells which must be added to the oxygen given off from the peroxide to give the total ogygen evolved* In the Warburg vessel were placed 1 ml* cells, 1 ml. phosphate buffer pH 7*0* 1*1 nil* 3aline and in the center cup was placed 0.2 ml. of twenty per cent HaOH to take up the carbon dioxide evolved. One-half milliliter of diluted hydrogen peroxide wqs placed in the side arm and added at equilibrium. For the control, to determine the oxygen uptake by the cells in the absence of peroxide, the vessels were set up as above except that no peroxide was present and 1.6 ml. of saline was added to keep the volume of liquid in the vessel at 5*6 ml* Preliminary trial showed the amount of peroxide used to be in excess. measurements were conducted at 55°C. for ^0 Respiration minutes* The results are shown on the following table* -56- Table 11. The Catalytic Activity of Micrococci Organism M. luteus C.mm. 0S uptake in absence of HS0S C.mm. 0S liberated in 50 Min*/Mgm. N 46.9 46.8 M. aurantiacus 17-9 17*6 20.1 M. cinnebareus M. freundenreichii 51.1 18.8 291.9 42.1 M. flavus 80.2 Examination for the Presence of Cytochrome Oxidase The cytochromes, or at least cytochrome-c, is oxidized by the enzyme cytochrome oxidase* This oxidase is a hemin compound and is inhibited by carbon monoxide and light and by cyanide* Stotz, Sidwell and Hogness (1938) found that cytochrome-c and the oxidase were involved in the oxidation of both hydroquinone and para—phenylenediamine, and that cytochrome—b, which does not oxidize hydroquinone, could function quite independently of this system in the oxidation of p-phenylenediamine. The ability of the Micrococci to oxidize hydroquinone and p—phenyl enediamine was determined as followst Xn the Marburg vessel were placed 1 ml. cells, 1 ml of phosphate buffer pH 7.05, 1.1 ml. saline and in the center cup was placed 0.2 ml. of twenty per cent NaOH to take up the carbon dioxide evolved. In the side arm was placed 0.5 ml. of hydroquinone or p-phenylenediamine 0.15 molar. -57- The p-phenylenediamine used was the hydrochloride which was adjusted to pH 6.95 immediately before use. It was found that hydroquinone showed a considerable rate of autoxidation at pH 7*05 and therefore this rate was determined as a blank in each experiment. Para-phenylenediamine, on the other hand, showed very little autoxidation* Hydroquinone was not oxidized by any of the organisms with the possible exception of M. freundenreichii which showed an endogenous respiration q02 of 5 5 .9 while in the presence of hydroquinone the q0 g was 54*5. In the presence of p-phenylenediamine an increased oxygen uptake was observed. This is shown in the following table. Table 12. The Oxygen uptake of Micro cocci in the Presence of Para-phenylenediamine. q0 s in absence of p-phenylenediamine M. luteus M. flavus M. aurantiacus M. cinnebareus M. freundenrei chii q0 s in presence of p-phenylenediamine 102.5 27*2 23.5 14.5 49*0 93.1 181.4 30*7 2 6 1 .3 M. auranticaus and M. freundenreichii 6 8 .2 were found to be most active in the oxidation of p-phenylenediamine while M. cinnebareus oxidized the compound very slowly. Since hydro quinone was not oxidized by any of the organisms, cytochrome-c -58- may be lacking in the cells* The rapid oxidation of p-phenylene— diamine might indicate that cytochrome—b is very active since Stotz et al* (193S) found that cytochrome-b could act independently of cytochrome—c and cytochrome oxidase in the oxidation of this compound. organisms with The absence of cytochrome-c would class these Staphylococcus aureus, albus and citreus and Sarcina aurantiaca which have been found by Frei at al (1934) to contain the cytochrome components a,b and d* In this connection Krampitz and Werkman (1941), however, found that Micrococcus lysodeikticus oxidized p-phenylenediamine but not hydroquinone* It was suggested that the cytochrome-c of this organism had a protein bearer differing from that of beef heart ( which oxidized both) which caused the potential of the former to be negative with respect to hydroquinone and hence inactive* Study of the Bacterial Pigment No principal differences have been found between the aarotenoids of higher and lower plants but those of the higher plants anly have been studied. For the algae only fucoxanthin is well characterized while with regard to the fungi and bacteria still less is known (Zechmeister, 1954)* Aside from the brief observations of Schrotter (1895) that the pigments of Serratia aurantiaca and Staphylococcus aureus show the solubility and lipocyan reaction of the barotenoids, -59- our knowledge regarding carotenoid producing species of bacteria is due to Zopf ( I899s->b; 1891, 1892) who has described the chromolipids in eight species of bacteria* The descriptions of Zopf point with certainty to carotenoids in the case of four species only, namely, Bacillus egregrium. Bacillus chrysoglia, Staphylococcus aureus and Sphaerotilus roseus. According to Zopf ji. egregrium and B* chrysoglia and Staphylococcus aureus produce a yellow carotenoid pigment characterized as follows: The cells produce an intense blue color with concentrated HsS04 (lipocyan reaction)* The pigment was slowly extracted by warm absolute alcohol and after extraction was soluble in alcohol, ether, chloroform, methyl alcohol, benzene and petroleum ether. The alcoholic solution showed two absorption bands, one covering the F line, the other between F and G* Sphaerotilus roseus, a red organism, gave a yellow-red alcoholic extract. The yellow poiment was water soluble while the red pigment was soluble in the fat solvents. In alcohol the latter pigment showed absorption bands at 492-474 mu and 456-442 mu* In 1925 Reader studied the carotenoid pigment of Sarcina aurantiaca • The cell suspension was saponified and the unsaponifiable fraction was extracted with petroleum ether, evaporated to dryness and taken up in light petroleum (B*P. 40-60°). This solution was passed through a chalk column and divided into fractions and examined spectroscopically* Two principal fractions were found, one of which appeared to be lycopin with absorption -60- bands between 509-490 mu, 477-458 mu, and 447-4^5 mu, while the other fraction possessed bands at 491-476mu and 456-440 mu and was believed to be carotene# Experimental Studies were conducted to determine whether the yellow pigment of M* luteus. the red pigment of M* cinnebareus. and the orange pigment of M.aurantiacus were carotenoid in nature# M* luteus, M. flavus and M* aurantiacus reacted positively to the lipocyan test with concentrated HgSO^# The yellow pigment of M. luteus was found to be soluble only in butyric acid, normal and secondary butyl alcohol* After extraction and reduction to dryness under reduced pressure the material was soluble only in butyric acid, normal and secondary butyl alcohol and glacial acetic acid. This material, after extraction and evaporation to dryness, no longer reacted positively to the lipocyan test* Tjjre absorption spectrum of the butyl alcohol extracted material was determined by means of the Oenco-Spectrophotometer* Before each reading the galvanometer was set at 100 with the secondary butyl alcohol alone and the reading of the solvent containing the pigment was determined* The bujpyl alcohol extract of M* luteus was found to possess absorption bands at 420 mu. and at 450 mu* After treatment of the cells with acetic acid a yellow extract was obtained with butyl alcohol, acetone or a mixture of -61- acetone and alcohol* This yellow extract obtained after treat— mant with acetic acid, however, possessed no absorption bands between yoO mu. and 680 mu* The orange pigment of M. aurantiacus possessed the same characteristics as the yellow pigment of M* luteus except that the absorption bands were located at 420 mu. and 490 mu* M. cinnebareus and M. freundenreichii did not react positively to the lipocyan test* Their pigments were found to be soluble in secondary butyl alcohol* The butyl alcohol extracted material of M. cinnebareus had a red color and possessed rather weak absorption bands at 510 mu* and 480 mu*, while the butyl alcohol extracted material of M. freundenreichii was light yellow in color and possessed rather strong absorption bands at 465 mu., 450 mu. and at 420 mu* -62- Summary The respiratory activities of Micrococcus luteus, Microcoecus flavus, Micrococcus aurantiacus, Micrococcus cinnebareus and Micrococcus freundenreichii were studied* With methylene blue as the hydrogen acceptor the compounds found to be most readily activated were raffinose, maltose, sucrose, glucose, ethyl alcohol, succinate, maleate and glutamate* When molecular oxygen was used as the hydrogen acceptor the compounds most readily activated were sucrose, maltose, glucose, ethyl alcohol, succinate, lactate, glutamate and asparagine. The oxygen uptake of most of the substrates was constant or decreased slightly with time, while some few of the substrates, mainly glutamate and dl-b-phenylalanine, showed an increasing oxidation rate* The influence on the cells of several respiratory inhibitors was determined. luteus and Micrococcus flavus c6ncentra.tion of 1.4x10 The dehydrogenases of Micrococcus were stimulated by a cyanide molar,.while the other organisms were inhibited by this concentration. The specific dehydrogenases were found to vary in their sensitivity toward inhibitors, those active against the amino acids being most susceptible in that they were strongly inhibited by sodium cyanide, sodium monochlor-acetate and sodium —5 azide in a concentration of 1*4x10 molar* -65- Using glucose as the representative substrate it was found that Miorococcus luteus was inhibited about JO per cent by a cyanide concentration of 1.4xl0”2 molar, while a 1.4x10“^ molar concentration inhibited respiration for a short time only after which an increasing rate was observed so that the total oxygen uptake at the end of one hour was identical with the uptake in the absence of inhibitor* A cyanide concentration of . -4 1*4x10 molar stimulated respiration about 40 per cent* Since the cell carriers operative in normal respiration were inhibited by the higher concentrations of cyanide, the ability of methylene blue to supplant these carriers was determined* Cyanide inhibition was generally decreased by methylene blue but was completely removed only in the case of Micro coccus flavus with sucrose as the substrate and Micrococcus aurantiacus with glucose as the substrate* Methylene blue when used alone was found to exert an influence upon the cells. Micrococcus luteus and Micrococcus flavus were stimulated by the presence of one milligram of methylene blue in the presence of all substrates except succinate, while the other organisms were inhibited by this amount* Micrococcus freundenreichii was stimulated by 0.5 milligram in the presence of all substrates escept succinate while Micrococcus aurantiacus and Micrococcus cinnebareus were inhibited by a concentration as low as 0*1 milligram. -64- Sodium monochlor-acetate in a concentration of —2 1*4x10 molar inhibited the oxidation of all substrates with the exception of glucose with which there was stimulation* Sodium azide was found to be a stronger inhibitor than sodium monochlor-acetate but the stimulation and inhibition paralleled, indicating the point of attack of both inhibitors to be the same* With the exception of Micrococcus freundenreichii * all the organisms possessed moderate polyphenol oxidase and catalase activity. Hydroquinone was not oxidized by any of the micrococci, while p-phenylenediamine was oxidized by all the organisms. This might indicate that cytochrome-b is abundant in the organisms or that cytochrome-c has a protein bearer which causes its potential to be negative with respect to hydroquinone and hence inactive. Micrococcus luteus, Micrococcus flavus and Micrococcus aurantiacus reacted positively to the lipocyan test with concentrated Hs S04 . After extraction with secondary butyl alcohol the absorption spectrum of the pigments was determined. The pigment of Micrococcus luteus possessed absorption bands at 420 mu and at 450 mu. The orange pigment of Micrococcus aurantiacus possessed absorption bands at 420 mu. and at 490 mu* Micrococcus cinnebareus and Micrococcus freundenreichii did not react positively to the lipocyan test. Their pigments were extracted with secondary butyl alcohol and possessed absorption bandsa at 510mu and 480 mu; and at 465mu, 450 mu and 420 mu, respectively. -65- Bibliography Blaschko,H. (1955) The machanism of catalase inhibitions. Biochem. J. 22: 2505-2512. Braun,H. and Vasarhelyi,J.v• (1952) Ueber die oxydrierenden und reduzierenden Pahigkeiten der Proteusbazillen und uber die Beeinflussung dieser Fermentwirkungen durch Antikorper und Antiseptika. Zentr. Bakt. Parasitenk.I Abt. Orig. 127a 105-111. Braun,H. and Wordehoff, Ph. (1955) Ueber die Oxydativen und reduzie­ renden Fermentwirkungen des Ruhrbazillus Flexner. Zbl. Bakteriol. Orig. 128: 50-81. Oallow, A.B. (1924) The oxygen uptake of bacteria. Biochem. J . L3: 507Oook, R.P. ( 1950) A comparison of the dehydrogenases produced by B. coli Oommunis in the presence of oxygen and methylene blue. Biochem. J* 24i 1558-1550. Cook,R.P., Haldane,J.B.S. and Mapson,L.W. (1951) The relationship between the respiratory catalysts of B. coli. Biochem. J. 25: 554-550. Das, N#B. (1957) Identity of lactic and malic cehydrogenases. Biochem.J. j51: 1116-1125* Ehrismann,0. (1955) Ueber die reduzierenden Wirkungem der Diphtherienbakterien. Zentr. Bakt. Parasitenk. I Abt. Orig. 155: 56^60. «1 Ehrismann, 0. (1957) UberDehydrierenden Leistungen von Mikrokken und Streptokokken. z.f. Hyg. und Infektsk. 119: 572-612. Elvehjem,C.A., ’ Wilson,P.V/. et al (1959) Respiratory enzymes. Burgess Publishing Go., Minneapolis. -66- Fabre,R. (1955) The hydrogen donors for the Staphylococci. Gompt. rend. 120: 6Q8-610. i/ Frei,W., Riedmuller,L. and Almasy,F. (1954) Atmungssystem der Bakterien. Gale, E.F. and Stephenson,M. (1959) Bio. Z. 274: 255-267. L-Malic dehydrogenase and codehydrogenases of Bact. coli. Gerard,R.W. (1951) 11 Uber Gytochrom und das Biochem. J. 1245-1256. Observations on the metabolism of Sarcina lutea. Biol. Bull. 60: 215-241. Green,D.E. and Stickland,L.H. (1954) Studies on reversible dehydrogen­ ase systems. I. The reversibility of the dehydrogenase system of Bacterium coli. Biochem. J. 28: 898-900. Green,D.E. and Stickland,L.H. and Tarr,H.L.A. (1954) Studies on reversible dehydrogenase systems. Ill Oarrier-linked reactions between isolated dehydrogenases. Green,D.E. and Brosteaux,J. (195^) animal tissues. Biochem. J. 28: 1812-1824. The lactic dehydrogenase of Biochem. J. Hawthorne,J•R. end Harrison,D.O. (1959) 1489-1508. Oytochrome-C as a carrier with the glucose dehydrogenase system. Biochem. J. ^5.: 1575-79* Johnson,A.A. and Green,J.R. (1950) Modified methyl red and sodium alizarin sulfonate indicators. J.Ind. and fing. Chem. (Analytical Edition) 2t 2-5. Krampitz,L.O. and Workman,C.H. (1941) Some properties of a bacterial cytochrome system (Abstract) Krebs,H.A. (1955) amino—acids. Bact. 4l: 21-22. Metabolism of amino-acids. III. Deamination of Biochem. J. 29 * 1620—1644. -67- Quastel, J»H. and Wooldridge,W.R. (1928) Some properties of* the dehydrogenating enzymes of bacteria, Biochem.J, 22: 689-702, Reader,V, (1925) bacteria. A note on the lipochromes present in certain Biochem. J. 1£: 10^9-1046. Schrotter,vH. (1895) Vorlaufige Mittheilung uber das Pigment von Sarcina aurantica und Staphylococcus pyogenes aureus. Oentr.Bakt.Parasitenk. (1) 18: 781-782. from Palmer,L.S., Oarotenoids and related pigments, Chemical Catalog Co. N.Y.,1922. Stotz,E., Sidwell,A.E. and Hogness,T.R. (1958) Thr role of the cytochromes in the action of // Indophenol Oxidase. S. Biol. Chem. 124: 755-744. Thunberg,T. (1918) Zur Kenntnis der Einwirkung tierischer Gewebe auf Methylenblau. Skand. Arch. Physiol. ^5: 165-195, Wooldridge, W.R.,Knox,R. and Glass,V. (1956) Variability in the activity of bacterial enzymes.I.The effect of the age of the culture. Biochem. J. j?0: 926-951* Yudkin,J. (1955) The dehydrogenase of Bact. coli. I.The effect of dilution: with a note on the existence of a coenzyme of glucose dehydrogenase. Biochem. J. 27: 1849—1858. Yudkin,J. (1957) The dehydrogenases of Bact. coli. III.The coenzyme of glucose dehydrogenase. Biochem. J. 51: 865-868. Zechmeister,L. (1954) Carotenoide, Jtilius Springer, Berlin, 1954. Zopf,W. (1889a) Ueber Pilzfarbstoffe. Botan. Ztg. 4£: 55-61, 69-81, 85-91. From Palmer,L.S. Oarotenoids and related pigments , Chemical Catalog Co., New York, 1922. -68- Zopf,W. (1889b) Ueber das microchemische Verhalten von Fettfarbstoffen und Fettfarbstoff-haltigen Organen. Z.wiss. Micros.6: 172-177-» from Palmer,L.S., Oarotenoids and related pigments, Chemical Catalog Co., New York, 1922. Zo£f,W. (1891) Ueber Aaescheidung von Fettfarbstoffen (Lipochromen) seiten gewisser Spaltpilze. Palmer, L.S., Catalog Co., Zopf,W. (1892) Ber. botan. Ges. Oarotenoids and related pigments, 22-28., from Chemical New York, 1922. Zur Kenntniss der Farbungsursachen niederer Organismen (Zweite Mittheilung) Beitr. Physiol. Morphol. niederer Organismen Z\ related pigments, , from Palmer,L.S., Chenical Catalog Co., Oarotenoids and New York, 1922.